Project Summary Genetic studies, first done in model eukaryotes and more recently in mammals, have revealed that homology-directed DNA repair (HDR) plays a critical role in the elimination of DNA double-strand breaks and in the preservation of stressed or injured DNA replication forks. HDR is reliant on the tumor suppressors BRCA1-BARD1, BRCA2, and PALB2, mutations in which cause breast, ovarian, and other cancers. Progress in understanding how these tumor suppressors help mediate HDR and how their mutational inactivation impacts upon genome integrity has been hampered by the challenge of purifying them for mechanistic studies. Our research team has overcome this challenge, which uniquely positions us to decipher the mechanisms by which these tumor suppressors support HDR. In conjunction with a growing research team in the NCI-designated Mays Cancer Center at the University of Texas Health Science Center at San Antonio and with external collaborators who are leaders in structural biology and single-molecule biophysics, we will dissect the underlying mechanisms of different stages of HDR, to specifically furnish insights regarding the roles of the aforementioned tumor suppressors therein. We will also pursue chemical screens and synthesis with the Cancer Prevention and Research Institute of Texas (CPRIT)-supported Center for Innovative Drug Discovery to develop inhibitors of HDR to use as a chemical biology tool and for preclinical studies. We are confident that our holistic approach to deciphering HDR mechanisms will provide the foundation for developing targeted cancer diagnostics and therapeutics.